PDF(Pubmed)
Abstract：
In eubacteria, cyclic di-GMP (c-di-GMP) signaling is involved in virulence, persistence, motility and generally orchestrates multicellular behavior in bacterial biofilms. Intracellular c-di-GMP levels are maintained by the opposing activities of diguanylate cyclases (DGCs) and cognate phosphodiesterases (PDEs). The c-di-GMP homeostasis in Mycobacterium smegmatis is supported by DcpA, a conserved, bifunctional protein with both DGC and PDE activities. DcpA is a multidomain protein whose GAF-GGDEF-EAL domains are arranged in tandem and are required for these two activities. To gain insight into how interactions among these three domains affect DcpA activity, here we studied its domain dynamics using real-time FRET. We demonstrate that substrate binding in DcpA results in domain movement that prompts a switch from an \"open\" to a \"closed\" conformation and alters its catalytic activity. We found that a single point mutation in the conserved EAL motif (E384A) results in complete loss of the PDE activity of the EAL domain and in a significant decrease in the DGC activity of the GGDEF domain. Structural analyses revealed multiple hydrophobic and aromatic residues around Cys579 that are necessary for proper DcpA folding and maintenance of the active conformation. On the basis of these observations and taking into account additional bioinformatics analysis of EAL domain-containing proteins, we identified a critical putatively conserved motif, GCXXXQGF, that plays an important role in c-di-GMP turnover. We conclude that a substrate-induced conformational switch involving movement of a loop containing a conserved motif in the bifunctional diguanylate cyclase-phosphodiesterase DcpA controls c-di-GMP turnover in M. smegmatis.
摘要：
在真细菌中，环状di-GMP（c-di-GMP）信号参与毒力，持久性,运动性，通常在细菌生物膜中协调多细胞行为。细胞内c-di-GMP水平由二鸟苷酸环化酶(DGC)和同源磷酸二酯酶(PDE)的相反活性维持。DcpA支持耻垢分枝杆菌的c-di-GMP稳态，一个保守的，具有DGC和PDE活性的双功能蛋白。DcpA是一种多结构域蛋白，其GAF-GGDEF-EAL结构域串联排列，是这两种活性所必需的。为了深入了解这三个域之间的相互作用如何影响DcpA活性，在这里，我们使用实时FRET研究了其域动力学。我们证明了DcpA中的底物结合会导致结构域移动，从而促使从“开放”构象转换为“封闭”构象并改变其催化活性。我们发现，保守的EAL基序（E384A）中的单点突变会导致EAL域的PDE活性完全丧失，并导致GGDEF域的DGC活性显着降低。结构分析揭示了Cys579周围的多个疏水性和芳香族残基，这是正确的DcpA折叠和维持活性构象所必需的。在这些观察的基础上，并考虑到对含EAL结构域蛋白的其他生物信息学分析，我们确定了一个关键的假定保守的基序，GCXXXQGF,这在c-di-GMP周转中起着重要作用。我们得出的结论是，底物诱导的构象转换涉及双功能二鸟苷酸环化酶-磷酸二酯酶DcpA中包含保守基序的环的移动，可控制耻垢分枝杆菌中的c-di-GMP周转。
